Molecular Identification Of Fallopia Multiflora By PCR-RFLP Based On ITS2 Sequences Analysis

ObjectiveFallopia multiflora Thunb. is a herbaceous perennial plant of the family Polygonaceae. Dry rhizome of F. multiflora, one of the famous traditional Chinese native medicine, possess very effective activities such as antioxidative, anti-aging, anti-inflammatory, anti-tumor, cholesterol-lowering and memory improving. Now the adulterants of F. multi flora are full of the market. Those adulterants, mostly including F. multiflora var.ciliinerve,Pteroxygonum giraldii, Cynanchum auriculatum. and Musa basjoo, jeopardise the safty and clinical efficacy of F. multiflora. The appearances of F.multiflora and its adulterants are similar, it is difficult for the general scientific research personnel to distinguish. F.multiflora, some of its closely related species and adulterants were investigated in this study. Based on the research and analysis of ITS2 sequences, we explored a new method to identify F. multiflora from its closely related species and adulterants.Methods1. Apply the kit, modified SDS method and modified CTAB method to extract the genomic DNA of the decoction pieces of F. multiflora.2. ITS2 regions of F. multiflora and its closely related species and adulterants were amplified, purified and sequenced. Sequences were assembled by Seqman and analysed by MEGA6.0, and NJ phylogenetic tree was constructed based on K2P model.3. Restriction enzyme analysis were carried out on F. multiflora and its closely related species and adulterants through Primer Premier 5.0 software. According to the differences of restriction map, a suitable endonuclease were detected to identify F. multiflora. Use slab gel electrophoresis and capillary gel electrophoresis to detect digestion products.4. Sequence comparative anlysis were carried out to find a stable variable site on F. multiflora and its closely related species and adulterants through Cluxtal X software. One specificity primers was designed based on the variable site. Use the primers to amplify all samples for further verify and use slab gel electrophoresis and capillary gel electrophoresis to detect digestion products.Results1. The kit isn’t appropriate for extracting the genomic DNA of F. multiflora which is made into decoction pieces. Both modified SDS method and modified CTAB method can extract F. multi flora gDNA, but modified CTAB is much better than modified SDS method.2. The ITS2 regions of F. multiflora exhibited obvious differences with its closely related species and adulterants. The intraspecific variation was less than the interspecific genetic distance. All samples of F. multiflora showed monophyletic on NJ tree, and they could be distinguished from its closely related species and adulterants.3. Based on the restriction enzyme analysis of ITS2 sequences, the endonuclease Nsb Ⅰ (Mst Ⅰ, Avi Ⅱ) were detected to identify F. multiflora from its closely related species and adulterants. The F. multiflora nrDNA ITS2 PCR products could be cleaved by Nsb I into two fragments of 238bp and 157bp, however, the ITS2 PCR products of its closely related species and adulterants could not be digested by Nsb I, the PCR-RFLP map of which exhibited only one band. The results of capillary gel electrophoresis are consistent with that of slab gel electrophoresis.4. An allele-specific PCR primer was designed according to 126G/C SNP mutation in ITS2 sequences. When the annealing temperature was 57℃, DNA from F. multiflora could be amplified 176bp whereas DNA from its closely related species and adulterants could not be amplified. The results of capillary gel electrophoresis are consistent with that of slab gel electrophoresis. ConelusionITS2 can be used as a effective marker for identifying F. multiflora and its closely related species and adulterants. Restriction enzyme analysis and sequence comparative anlysis were carried out on ITS2 sequences of F. multiflora and its closely related species and adulterants, which suggested that PCR-RFLP and allele-specif ic PCR could be used to identify the traditional Chinese medicine F. multiflora.It is the first time to utilize capillary gel electrophoresis on PCR-RFLP and allele-specific PCR identification of F. multiflora, the result of which is consistent with agarose electrophoresis. However, capillary gel electrophoresis has formidable strengths such as higher sensitivity, clear chromatograms, quantitative and qualitative available, rapidity, clear and pollution-free, automated analysis and synchronous processing data. Thus, capillary gel electrophoresis has great potential value for substituting slab gel electrophoresis to analyse DNA.